Frontiers in Drug Design & Discovery: Volume 10

By Atta-ur Rahman and M. Iqbal Choudhary

Frontiers in Drug Design and Discovery is a book series devoted to publishing the latest and the most important advances in drug design and discovery. Eminent scientists have contributed chapters focused on all areas of rational drug design and drug discovery including medicinal chemistry, in-silico drug design, combinatorial chemistry, high-throughput screening, drug targets, and structure-activity relationships. This book series should prove to be of interest to all pharmaceutical scientists who are involved in research in drug design and discovery and who wish to keep abreast of rapid and important developments in the field.

The tenth volume of this series brings together reviews covering topics related to the treatment of neoplasms, systems biology, respiratory diseases among others.

Topics included in this volume are:

- Recombinant Protein Production: from Bench to Biopharming

- Plant Virus Nanoparticles and Virus like Particles (VLPs): Applications in Medicine

- MAO Inhibitory Activity Of 4, 5-Dihydro-1 HPyrazole Derivatives: A Platform To Design Novel Antidepressants

- Flavonoids Antagonize Effects of Alcohol in Cultured Hippocampal Neurons: A Drug Discovery Study

- Hybrid Smart Materials for Topical Drug Delivery: Application of Scaffolds

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Feb 3, 2021
• ISBN: 9789811421563

Atta-ur Rahman

Atta-ur-Rahman, Professor Emeritus, International Center for Chemical and Biological Sciences (H. E. J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research), University of Karachi, Pakistan, was the Pakistan Federal Minister for Science and Technology (2000-2002), Federal Minister of Education (2002), and Chairman of the Higher Education Commission with the status of a Federal Minister from 2002-2008. He is a Fellow of the Royal Society of London (FRS) and an UNESCO Science Laureate. He is a leading scientist with more than 1283 publications in several fields of organic chemistry.

Book preview

Recombinant Protein Production: from Bench to Biopharming

Rais A. Ansari¹, *, Shakil A. Saghir², ³, Rebecca Torisky², Kazim Husain⁴

¹ Department of Pharmaceutical Sciences, College of Pharmacy, Health Professions Division, Nova Southeastern University, 3200 S University Drive, Fort Lauderdale, FL 33328, USA

² Scotts Miracle-Gro, 14111 Scottslawn Road, Marysville, OH 43041, USA

³ Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan

⁴ Department of Gastrointestinal Oncology, Moffitt Cancer Center and Research Institute, 12902 USF Magnolia Drive, Tampa, FL 33612, USA

Abstract

The needs for purified proteins in modern medicine, research and industrial application are immense and production of proteins using recombinant technology offers solutions; proteins are used in simple laboratory experiments like protein-protein and protein-DNA interactions and in diagnostic, therapeutic and industrial applications. Some examples of the application of purified recombinant proteins for the treatment of diseases include clotting factors (Factor VIII and IX) for the treatment of hemophilia, insulin-dependent diabetes, and adenosine deaminase for severely compromised immune disease. Recently, human monoclonal antibodies, like anti-tumor necrosis factor-α (Adalimumab) for the treatment of rheumatoid arthritis and Repatha (proprotein convertase subtilisin kexin type 9 or PCSK9) inhibitor antibody for the treatment of and reduction in the risk of myocardial infarction, stroke and revascularization of coronary artery diseases, are produced using protein overexpression methodology described in this chapter. Use of recombinant protein technologies has enabled industries to produce proteins of human significance at a tremendous pace. Production of therapeutic proteins at large scale for millions of individuals to treat diseases is one of the essential needs of mankind. From simple proteins like albumin, growth factors, cytokines, viral vaccines and human monoclonal antibodies, all are being produced utilizing the recombinant protein expression technology and purification processes, whether in a laboratory or biopharming scale in microorganisms, animals and/or plants. This chapter summarizes various recombinant expression systems and their pharmaceutical applications.

Keywords: Adenovirus Expression System, Baculovirus-Mediated Expression System, Biopharming, CHO Expression System, Eukaryotic Expression System, Gene of Interest, History of Biopharming, Mammalian Expression System, Possible Contaminants in Expression Systems, Prokaryotic Expression System, Protein Expression System, Recombinant Proteins, Shuttle Vector, Vaccinia Virus Expression System, Yeast Expression System.

---

* Corresponding author Rais A Ansari: Department of Pharmaceutical Sciences, College of Pharmacy, Health Professions Division, Nova Southeastern University, 3200 S University Drive, Fort Lauderdale, FL 33328, USA; Tel/Fax: [phone]; E-mail: [e-mail]

INTRODUCTION

Human beings use proteins or smaller peptides in different ways, which could be enzymes added to soap or use of growth hormone for the treatment of pituitary-driven dwarfism. Such proteins can be obtained from various sources. However, yields were previously low and the cost of purifying them was quite high, limiting their production and use. Advancements in the area of recombinant protein production has changed the trend making the yields much higher and the cost much lower, allowing the production of such proteins on industrial scale, opening the door for the treatment of multiple diseases and disorders discussed in this chapter. For example, bovine and porcine insulins had been used for the treatment of insulin-dependent diabetes, and they have now been replaced by human insulin produced in Escherichia coli using the recombinant technology. This technology has also enabled us to avoid potential contaminations from pathogens of animal origin, like viruses. By using recombinant protein technology, we can overexpress desired proteins and biopharm them using microorganisms, animals, and/or plants.

BASIC EXPRESSION CONCEPT

In almost all systems for expression of recombinant proteins, either plasmids carry or are used to create the expression viruses with a gene of interest (GOI) which is driven by a promoter from another gene (a heterologous system) which is active in the organism wherein the protein is being expressed. Isolation of proteins and their purity remains the issue; therefore, an affinity tag is added to either the amino-terminal (N-terminal) or carboxyl-terminal (C-terminal) of the proteins. The tags serve for isolation and purification of the protein and read in frame of the GOI. In order to add the tag at either N- or C-terminal, 4-6 uncharged amino acids are used between tag and protein (Fig. 1). Usually, an endopeptidase site is present between the GOI and the tag so that tag can be removed enzymatically. For a majority of tags (e.g., glutathione-S-transferase [GST], maltose binding protein [MBP], chitin, strep-tag, polyarginine [p-Arg], and 6xhistidines [6xHis]) affinity resins are used, while for other tags (e.g., small ubiquitin-related modifier [SUMO], FLAG-tag, c-myc peptide, and 1D4 epitope) (for the list of affinity tags and acronyms, see (Table 1) antibody-resin affinity columns are employed for purification [1, 2]. A single tag, either at the N- or C-terminal, is not efficient for obtaining sufficiently quality proteins, therefore, dual tags (one at the N- and the other at the C-terminal or in tandem) are routinely used to further enhance the purity of the proteins. A single step 6xHis tag GOI purification using nickel-nitrilotriacetic acid (Ni-NTA) or other metal-containing resins does not produce a satisfactory purified protein. With dual fusion, one additional affinity purification, following 6xHis-affinity purification, removes the contaminating proteins. To increase the purification further, the dual affinity purified sample is subjected to high pressure liquid chromatography (HPLC) or specific protocols developed for the purification of that GOI.

Fig. (1))

The basic concept of recombinant proteins and its expression system.

Table 1 List of the affinity/tag.

In prokaryotic systems, plasmids directly serve for the expression of GOI in bacteria while viruses (e.g., baculovirus, adenovirus and vaccinia virus) express recombinant proteins by directly infecting the cells. These recombinant viruses when infected to appropriate cells produce the GOI with tag(s). The promoter-derived expression of GOI in a specific cell or organism is either a promiscuous promoter or the promoter of that species-specific gene. Therefore in baculovirus, the polyhedrin gene promoter or the p10 gene promoter which derives expression of the capsid protein is utilized [3, 4]. Similarly, GAL (galactose - inducible gene) promoters which are active in yeast and promoters of other genes like alcohol dehydrogenase (ADH) and glyceraldehyde-3 phosphate dehydrogenase (G3PDH) are used for expression in yeast [5, 6]. Specific viral promoters (T7 and SP6) are also used for driving the gene expression of the GOI [7, 8]. In certain prokaryotic (pET) systems, T7 RNA polymerase remains under the ptac promoter and when activated by isopropyl β-D-1-thiogalactopyranoside (IPTG) drives the expression of T7 RNA polymerase, which binds to T7 promoter for driving the expression of the GOI (http://www.emdmillipore.com/US/en/product /pET-Expression-System-28- Novagen).

The mammalian system includes viruses which infect mammalian cells. The promoters of these viruses utilize the mammalian machinery for replication and production of proteins for viral replication. The two most significant viral expression systems, adenovirus and vaccinia viruses, utilize the cytomegalovirus major immediate early promoter (CMV-MIEP) which is active in most cell types and used for expressing of recombinant proteins [9]. In addition, vaccinia virus utilizes T7 RNA polymerase in trans for the expression of recombinant proteins. For vaccinia virus-based expression, the vaccinia virus T7 RNA polymerase is expressed from vaccinia virus from its promoter (p7.5) which drives the expression of GOI from T7 promoter. The recombinant vaccinia virus with GOI under T7 promoter is produced in thymidine kinase (TK-) cell line and the recombination is achieved at the TK locus which is knocked off in the TK-cells. In order to carry recombination at TK site, the GOI is cloned flanked on both sides with TK gene sequences utilizing the plasmid pGS53. Plasmids are also used for producing a stably transfected cell line deriving GOI expression from CMV-MIEP for the production of recombinant proteins in various mammalian cell lines including Chinese hamster ovary cell line (CHO). In addition to CMV-MIEP, other promoters have also been used [10].

EXPRESSION SYSTEMS

Prokaryotic Expression System

A number of bacterial hosts have been utilized for heterologous protein expression and E. coli and Bacillus subtilis are the two important and most commonly used prokaryotic expression systems. E. coli is the most commonly used prokaryote for the expression and production of recombinant proteins from its gene promoters [11, 12]. Prokaryotes lack post translational modification of proteins. Proteins which lack posttranslational modification, like glycosylation and sumoylation, and are cytosolic in nature, with molecular weight less than 60 kd are easily expressed in a prokaryotic system, especially in E. coli. Proteins which are post- translationally modified require expression using eukaryotic and mammalian expression systems. When a prokaryotic system is used for the expression of proteins that require glycosylation, glycosylating enzymes must also be expressed in a prokaryote like E. coli. However, when the activity of the protein is linked to glycosylation, inactive proteins may be produced due to differing nature of glycans in prokaryotes. Membrane-bound proteins are not good candidates for expression in prokaryotes due to the association of GOI with lipids, since plasma membranes are absent in E. coli. A list of prokaryotic expression systems utilizing various promoters are summarized in Table 2.

Table 2 List of various promoter systems of prokaryotes and their sources.

A typical prokaryotic expression system involves a promoter of a prokaryotic gene (ptac is a fusion promoter of ULV5 and tryp operon) [13] which is regulated by the addition of a lactose derivative, IPTG. IPTG cannot be metabolized (non-hydrolyzable) by E. coli [13] or repressor deactivated by heat when the culturing condition of the heat-inducible promoter is switched from 37oC to 42oC to induce protein expression from lamda phage (λPL) promoter. The λPL promoter utilizes the thermosensitive, λcI repressor protein (cI857) which cannot fold naturally at 42oC and therefore cannot bind to the promoter to repress transcription [14]. Expression from the ptac promoter using IPTG results in large amounts of IPTG discharge in the environment; therefore, heat inducible promoters are considered a better choice. In a different set of conditions, when the promoters are from cold shock protein, expression is achieved by reducing temperature. The ptac promoter remains under the control of up-mutant (lacIq) product which is made operational by adding IPTG. The single gene lacI is present in genome and its product cannot efficiently regulate the multi-copy plasmid under the control of ptac. Therefore, lacI is mutated to produce additional copies of lacIq to accommodate the inhibition of multiple copy plasmids deriving the expression from ptac promoter. It is evident that in prokaryotes, lacIq plays a significant role in the expression of recombinant proteins [15] (Fig. 2).

The pQE-series of plasmids (Qiagen [16], now Life Sciences) lack the lac repressor upmutant (lacIq) on the plasmid which is always multiple copy and cannot be completely inhibited from lacIq gene product which is present in the E. coli genome. The E. coli strain DH5α lacks lacIq in the genome and when used for cloning GOI in pQE- series plasmids results in clones where the GOI remains constitutively active causing death of positive clones at the expense of excessive expression of GOI (personal observation). Therefore, for GOI to express as 6xHis-fusion using pQE-series plasmids, E. coli strain containing lacIq in the genome (Sure cells) is used for in-frame cloning.

Fig. (2))

Mechanism of lac operon based protein expression..

The pET system developed by Novagen is a powerful system that ensures no leaky expression of the recombinant protein. It utilizes a 20-nucleotide long T7 RNA polymerase promoter which cannot be recognized by E. coli RNA polymerase. Therefore, in the absence of T7 RNA polymerase, no protein is expressed. A T7 RNA polymerase of DE3 phage is expressed from lacUV5 promoter after induction with IPTG. In the absence of IPTG, T7 RNA polymerase is not expressed due to inhibition by lacIq; the promoter upmutant is ten times more effective in carrying out the inhibition. To avoid leaky expression of pET, this system uses T7 lysozyme that inhibits T7 RNA polymerase, which is achieved by introducing another plasmid, pLysS or pLysE. These plasmids carry the T7 lysozyme gene either in silent (pLysS) or expressed (pLysE) orientations in relation to tetracycline responsive promoter (Tc) [17].

The glutathione S-transferase (GST) fusion (pGEX-series) and maltose binding protein fusion (MBP; pMalp2 or pMalc2 series) plasmid (Invitrogen [18], now Thermo Fisher Scientific) carry lacIq on the plasmid inhibiting the leaky expression and therefore DH5α is used for cloning the GOI, for both GST and MBP fusions. The MBP fusions are better than GST due to the solubility of MBP [19, 20] than many other fusions proteins (e.g., 6xHis and FLAG-tag fusion proteins). Many other fusion proteins such as metal binding protein, calcium calmodulin binding protein (CaBP), chitin binding protein (CBP) and acyl carrier binding protein (ACP) are also available [21]. Prokaryotic expression systems are easier than other systems as it requires simple culture conditions (e.g., media, additives), low cost, easy scalability, and offers large scale production of recombinant protein in a short period of time (e.g., doubling time of E. coli is 20 min). One of the greatest successes of utilizing a prokaryotic system to express proteins is the production of recombinant insulin by Elli Lilly [22].

Eukaryotic Expression System

Shuttle Vectors

A eukaryotic protein expression system involves expression of GOI from a eukaryotic/mammalian promoter. The activity of a promoter is very important as the transcription factors have to find the promoter to drive the expression of GOI. Often the GOI is cloned into a plasmid which is replicated in E. coli and later used to stably transfect cells and/or cell lines, like human embryonic kidney-293 (HEK293), human liver (HepG2), Chinese hamster ovary (CHO), HeLa, and baby hamster kidney (BHK) cells. In these situations, a eukaryotic origin of replication which is mostly of viral origin for replication is added to the plasmid. For selecting stably transfected cell lines (e.g., HEK293, HepG2, CHO), a eukaryotic antibiotic resistance gene (e.g., Hygromycin, G418, Puromycin, Zeocin) is also cloned onto the plasmid. Such a plasmid is referred to as "Shuttle vector" and is replicated and selected in prokaryotes and eukaryotes. The majority of plasmids used to express the protein in eukaryotic and mammalian cells are shuttle vectors. For lower eukaryotes like yeast, the origin of replication is an autonomously replicating sequence (ARS) derived from the yeast chromosome. The features of shuttle vectors are summarized in the Fig. (3). In the proceeding sections, the eukaryotic and mammalian system will be described for the expression of GOI.

Fig. (3))

Features of a shuttle vector.

Yeast Expression System

Primitive eukaryotes which are convenient to grow are utilized for the production of recombinant proteins. Two yeast systems (Saccharomyces cereviceae and Pichia pastoris) are on the forefront of eukaryotic expression systems. In primitive eukaryotes, proteins can be glycosylated and secreted which is not observed in prokaryotes. The only problem with glycosylation in S. cereviceae and P. pastoris expression systems is the nature of glycan moiety. For example, in plants and yeasts, glycosylation is of a mannose type whereas animal and human proteins contain glycosylation of galactosamine, sialic acid and glucose. When glycosylation does not affect activity of expressed proteins, lower eukaryotes are used for the expression of human/animal glycosylated proteins. Since the activity of antitrypsin is not affected due to the nature of glycosylation, it is expressed in lower eukaryotes [23]. The cost of expression of proteins in the lower eukaryotes is higher than in the prokaryotes. However, lower eukaryotes can be modified to carry glycosylation-specific genes of mammalian proteins (i.e., humanized proteins) to produce humanized glycoproteins like antibodies [24].

Like prokaryotes, yeast expression systems are also created by cloning GOI into plasmids. Also, as with prokaryotes, yeast-expression plasmids require sequences for maintenance either episomally at the origin of replication or integrated into chromosomes. The plasmids also carry a strong promoter and biomarkers for prokaryotes and eukaryotes for the selection of clones. In yeast expression systems, in addition to integrated plasmids (YIp) and episomal plasmids (YEp), centromeric plasmids (YCp) are also available for expression [25]. The YIp vectors do not replicate autonomously, rather integrate into the chromosome at low frequency providing fewer copies (up to 20) of the GOIs [25]. The YEp vectors carry the natural 2 µm episomal origin of replication providing the ability of plasmids to replicate independently (https://blog.addgene.org). Although YEp plasmids are capable of replicating episomally in the culture, some yeast cells lose the plasmid. Due to loss of YEp plasmid from yeast, large-scale expression of recombinant proteins is not recommended using the YEp plasmid system. Like YEp plasmids, YCp plasmids contain ARS and centromere sequences. YCp-based plasmids tend to be of low copy in yeast cells.

As with prokaryotes, selection of promoters for yeast expression system is achieved by using compatible promoters. In the case of yeast, an inducible promoter such as alcohol dehydrogenase -2 (ADH2), Sucrose -2 (SUC2) and constitutive gene promoter(s) like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are used. In order to select transformed clones, auxotrophic gene supplementation is used, which is being supplemented when yeast is not transformed and used for selection. The yeast expression systems are shown in Fig. (4).

Fig. (4))

Expression plasmids for Yeast.

CHO Expression System

For therapeutic proteins to be produced in active form, proper posttranslational modifications and folding are required. Often, it includes posttranslational modification in the form of glycosylation, either N-linked to asparagine or O-linked to serine/threonine. Glycoproteins are usually produced in mammalian cells, whereas E. coli and other prokaryotic systems are deficient in glycosylation. However, attempts to provide specific enzymes for glycosylation into a prokaryotic system to create glycoproteins must take into account their potential adverse biological properties. Therefore, eukaryotic cells derived from rodents and humans (e.g., NIH 3T3, CHO, BHK, HeLa and HepG2) are frequently used for recombinant protein expression from heterologous systems. Despite the availability of many cell lines, the majority of recombinant proteins are produced in CHO cells. The recombinant proteins obtained from CHO cells are biologically active for therapeutic purposes. Additionally, deadly viruses like HIV, influenza, polio, herpes and measles do not replicated in CHO cells, eliminating the possibility of recombinant protein contamination with aforementioned viruses and possible human exposure. A large number of biomoleucles, such as hormones, enzymes and antibodies are produced in CHO cells and cleared for use by US Food and Drug Administration (FDA) [26, 27]. The current global annual sales of biologics produced using CHO cells alone exceed US$30 billion [26].

Although CHO cells are adherent cells, they have been adopted for suspension and thus are scaled up to 10,000 liter capacity for industrial-scale recombinant protein production. The GOI is carried by a shuttle vector which provides both prokaryotic and eukaryotic origins of replication and selection biomarkers for the selection in prokaryotic and eukaryotic (i.e., CHO cells) systems. For a simple GOI expression, CMV-MIEP is used, but a CHO mutant requires dihydrofolate for its growth utilizing expression of GOI from the promoter of dihydrofolate reductase (DHFR) gene [28]. Two DHFR-deficient mutants (DXB11 and DG44) were created from proline auxotroph by Chasin and co-workers and are commonly used today [29-31]. The DHFR mutant is transfected with plasmid carrying DHFR gene and GOI under DHFR promoter and grown under the inhibitor, methotrexate (MTX). Inhibition of DHFR metabolism to tetrahydrofolate by methotrexate puts pressure on cell survival inducing the multiplication of DHFR gene which in turns increases the copy number of GOI. This allows increase in copy number of GOI and thus increased expression of recombinant protein as shown in Fig. (5).

Baculovirus-Mediated Expression

Baculoviruses are important for the expression of recombinant proteins especially of eukaryotic origin needing posttranslational modification [32]. The proteins are secreted and glycosylated; however, since the proteins are expressed in insect cells, the nature of glycosylation is different than mammalian glycosylation. The baculoviruses are of two types; Autographa california (alfalfa looper) nuclear polyhedrosis virus (AcNPV) and Bombyx mori (silkworm) nuclear polyhedrosis virus (BmNPV) which have a ~130 kb genome size. Large sections of DNA (up to 30 kb) are inserted into baculovirus expressing genes from late promoters (p10 or polyhedrin gene) which encode architectural proteins. Multiple genes can be expressed in insect cells by infecting them with multiple baculovirus and determining each viral titer in the system.

Fig. (5))

A schematic presentation of the expression of recombinant protein in CHO cells.

Baculoviruses are produced using a recombination process. Traditionally, the GOI is cloned into a transfer vector which is propagated in E. coli. The transfer vector with GOI is transfected into competent DH10BAC E. coli cells that contains a bacmid and helper plasmid. The helper plasmid provides the necessary enzymes for recombination of donor plasmid carrying GOI under the p10 or polyhedrin promoter flanked with recombination sequences to recombine with bacmid. The bacmid is purified and transfected to insect cells. The baculovirus bud out within 3-5 days. These viruses are again used to infect insect cells for further propagation and growth of viruses (Fig. 6). The plaque forming unit (pfu) activity of viruses are determined for infection of insect cells. A pfu of 3-5/cell is used for expression of recombinant proteins.

The most common cell line used for baculovirus expression system, SF9 cells, is a clonal isolate of the cell line IPLB-SF21-AE of Spodoptera frugiperda (fall armyworm). SF9 was originally established from ovarian tissue [13]. In addition to SF9 cells, SF21, Tn-368 and High-Five™ BTI-TN-5B1-4 are also used for the expression of recombinant proteins [33]. There are various claims of better expression of recombinant protein by one or the other cell lines and individual investigators are required to determine the best cell line for the expression of the protein of interest.

Fig. (6))

Baculovirus based expression of GOI.

Because of the nature of infection caused by baculoviruses, there is no possibility of disease(s) from baculovirus infection to humans. Baculovirus-produced recombinant proteins contain high mannose content and therefore the mammalian glycoproteins when expressed in baculovirus may differ in activity. Besides differing in nature of glycosylation, severe acute respiratory syndrome (SARS) virus glycoprotein is produced by the baculovirus expression system [34]. In addition, baculovirus possesses limited capacity to process pro-proteins into active protein as they lack pro-convertase, therefore preprocessing of proteins may be challenging.

Mammalian Expression

Adenovirus Expression System

Several adenoviral expression systems are available which are generated either by cloning or using a process of recombination. The GOI is cloned into a shuttle vector. This shuttle vector carries the GOI to an adenoviral backbone by the process of subcloning or by recombination. The pAdenoX vector is used for the replication of incompetent adenovirus containing and expressing the GOIs. The GOI expression is